Background This thesis investigated the efficacy and safety of Bacopa monnieri in improving memory in healthy Australians over the age of 55-years. A review of the literature showed that memory impairment and dementia are increasingly prevalent in the current demographic climate of an ageing population. As well as the pathological cognitive loss of neurodegenerative disease, many older persons are experiencing memory loss as part of the physiological process of ageing. Bacopa monnieri is a herbal medicine used since antiquity in the traditional Ayurvedic medical system of India for its cognitive enhancing effects. A number of pre-clinical and clinical studies support this traditional usage. Laboratory studies have demonstrated antioxidant and cholinergic actions in the brain as well as improved memory and cognitive performance in animal models. Human trials of Bacopa have also demonstrated improved memory performance. Some of these trials are limited by methodological flaws such as lack of blinding, small sample sizes, or use of outcome measurements which are not well validated. However, a small number of well designed human trials provide evidence for efficacy in cognitive and memory performance improvement. The current study was employed to extend on previous findings by assessing the efficacy and safety of Bacopa in the aged population specifically, as it is in this population that memory impairment becomes apparent. Aims 1. To assess the efficacy of Bacopa monnieri in improving memory in healthy Australians over the age of 55-years. 2. To assess whether the use of Bacopa is associated with side-effects Design A 12-week, randomised, double-blind, placebo-controlled, parallel group clinical trial. Participants Participants were self selected from the general population. They were aged 55-years or over at the commencement of the trial. Participants were without dementia, depression or other serious health conditions and did not use psychotropic medications. Intervention Participants were randomised to one of two treatment conditions, either a tableted extract of Bacopa monnieri called Bacomind™ (300mg/day, standardised to contain at least 40% bacosides), or an identical placebo. Participants attended three clinical evaluations: the first an initial screening session, the second a baseline evaluation of neuropsychological function and subjective memory performance at the commencement of the trial and the third, an end-of-trial outcome evaluation at 12-weeks, during which neuropsychological function and subjective memory performance were again assessed along with side-effects and study compliance. Primary Outcome Measures Rey Auditory Verbal Learning Test (AVLT), Rey-Osterrieth Complex Figure Test (CFT), Memory Complaint Questionnaire (MAC-Q), and Trail Making Test (TMT) Results From 136 people who elected to participate, 103 people met study entry criteria and 98 of these commenced the trial. Of these, 81 participants completed the trial and provided evaluable data for the end point analysis. Bacopa monnieri versus placebo significantly improved verbal learning as well as delayed recall as measured by the AVLT (p<.05). Though improvements were noted in the CFT, MAC-Q and TMT, there were no significant differences between placebo and active groups found for these tests. The Bacopa group reported a higher incidence of gastro-intestinal (GIT) side-effects than the placebo group, these predominantly being increased stool frequency, abdominal cramps and nausea. No other significant adverse effects were found. Conclusions A clinical trial was carried out to assess the effects of 12-weeks administration of Bacopa monnieri (300mg/day) on memory performance in people over the age of 55-years. Primary outcome measures were well validated neuropsychological tests that objectively measured verbal and visual memory and a memory complaint questionnaire that measured subjective memory complaints. The results demonstrated that Bacopa significantly improved memory acquisition and retention in older Australians. This concurs with findings from previous human and animal studies, as well as supports traditional Ayurvedic claims and uses. The beneficial effects on memory observed may be due to previously demonstrated antioxidant and cholinergic effects of the herb on the central nervous system. The use of Bacopa was associated with GIT side-effects, particularly increased bowel movements, nausea and abdominal cramping, findings infrequently reported previously. Possible explanations for these side-effects include GIT irritation by the saponin constituents of the herb, or cholinergic stimulation of autonomic and motor responses in the GIT, or a combination of both of these factors. The side-effects observed in the current study provide supportive evidence that Bacopa may increase cholinergic activity in humans. A worthwhile future extension of the current study would be to assess whether the finding of Bacopa’s efficacy for improving memory performance is replicable in populations with either mild cognitive impairment or early dementia.

Soy contains many bioactive molecules known to elicit anti-cancer effects. One such peptide, Lunasin, has been shown to selectively act on newly transformed cells while having no cytotoxic effect on non-tumorigenic or established cancer cell lines. In this study we attempt to understand the developmental effects of Lunasin overexpression in vivo and create reagents that will help us understand Lunasin’s anti tumorigenic effects in an intact organism. cDNA encoding lunasin and EGFP-lunasin were cloned into pUAST and microinjected into Drosophila embryos. Tissue-specific overexpression of EGFP-Lun in the resulting transgenic lines was accomplished by crossing transgenics to various GAL4 driver lines. Progeny were assessed for phenotypic alterations and no phenotypic abnormalities were observed in tissues expressing EGFP-Lunasin, supporting current studies that show Lunasin does not affect normal cells. Previous studies have localized Lunasin to the nuclear compartment. To test if this was the case for EGFP-Lun, subcellular localization of EGFP-Lun was determined via fluorescence microscopy. Salivary glands from EGFP-Lun expressing individuals were dissected, fixed, and mounted in Vectashield® with the nuclear stain, DAPI. Our results demonstrate that EGFP-Lun, like native Lunasin, is localized to the nucleus. Eight transgenic lines were mapped to specific chromosomes and EGFP-Lun transgenic line GEJ1-L2 was balanced in preparation for use in tumor suppression studies. In summary, we have created and characterized transgenic flies capable of overexpressing Lunasin under the control of the GAL4/UAS system. Localization of EGFP-Lunasin to the nucleus and data on the phenotypic consequence of its overexpression in flies is presented. Finally, reagents created as part of this thesis will aid experiments aimed at understanding the effects of Lunasin on benign and invasive tumors.

Ethanol causes neurotoxicity via several mechanisms at different points in the cycle of dependence, including neuroinflammation and oxidative stress during ethanol exposure as well as excitotoxicity during ethanol withdrawal. The primary therapeutic implication is that ethanol-induced neurotoxicity requires multifunctional pharmacotherapies which reduce all mechanisms. Using an innovative pharmacological high throughput screening method on a large plant extract library we discovered flavonoids with alpha7 nicotinic acetylcholine receptor (nAChR) activity. In addition to their well-known anti-inflammatory and antioxidant properties, this novel activity means they can potentially reduce excitotoxicity and therefore makes them ideal for inhibition of ethanol-induced neurotoxicity. Rhamnetin, the candidate compound, was first found to inhibit lipopolysaccharide induced inflammation in immortalized BV2 microglia, in part, via alpha7 nAChRs. We then established an in vitro model of ethanol induced-neurotoxicity using organotypic hippocampal slice cultures which incorporated both neuroinflammatory and excitotoxic components. Neuroinflammation enhanced excitotoxicity under control conditions but the reverse was observed during ethanol withdrawal. Both mechanisms are important but their interaction is not simple. Finally, rhamnetin was evaluated in this model and found to reduce neuroinflammation and excitotoxicity associated with ethanol withdrawal. In conclusion, the studies herein provide strong evidence for alpha7 nAChRs selective flavonoids as potential pharmacotherapies for the treatment of ethanol-induced neurotoxicity and further implicate neuroinflammation, excitotoxicity, and their interaction as critical mechanisms in this pathology.

Background This thesis investigated the efficacy and safety of Bacopa monnieri in improving memory in healthy Australians over the age of 55-years. A review of the literature showed that memory impairment and dementia are increasingly prevalent in the current demographic climate of an ageing population. As well as the pathological cognitive loss of neurodegenerative disease, many older persons are experiencing memory loss as part of the physiological process of ageing. Bacopa monnieri is a herbal medicine used since antiquity in the traditional Ayurvedic medical system of India for its cognitive enhancing effects. A number of pre-clinical and clinical studies support this traditional usage. Laboratory studies have demonstrated antioxidant and cholinergic actions in the brain as well as improved memory and cognitive performance in animal models. Human trials of Bacopa have also demonstrated improved memory performance. Some of these trials are limited by methodological flaws such as lack of blinding, small sample sizes, or use of outcome measurements which are not well validated. However, a small number of well designed human trials provide evidence for efficacy in cognitive and memory performance improvement. The current study was employed to extend on previous findings by assessing the efficacy and safety of Bacopa in the aged population specifically, as it is in this population that memory impairment becomes apparent. Aims 1. To assess the efficacy of Bacopa monnieri in improving memory in healthy Australians over the age of 55-years. 2. To assess whether the use of Bacopa is associated with side-effects Design A 12-week, randomised, double-blind, placebo-controlled, parallel group clinical trial. Participants Participants were self selected from the general population. They were aged 55-years or over at the commencement of the trial. Participants were without dementia, depression or other serious health conditions and did not use psychotropic medications. Intervention Participants were randomised to one of two treatment conditions, either a tableted extract of Bacopa monnieri called Bacomind™ (300mg/day, standardised to contain at least 40% bacosides), or an identical placebo. Participants attended three clinical evaluations: the first an initial screening session, the second a baseline evaluation of neuropsychological function and subjective memory performance at the commencement of the trial and the third, an end-of-trial outcome evaluation at 12-weeks, during which neuropsychological function and subjective memory performance were again assessed along with side-effects and study compliance. Primary Outcome Measures Rey Auditory Verbal Learning Test (AVLT), Rey-Osterrieth Complex Figure Test (CFT), Memory Complaint Questionnaire (MAC-Q), and Trail Making Test (TMT) Results From 136 people who elected to participate, 103 people met study entry criteria and 98 of these commenced the trial. Of these, 81 participants completed the trial and provided evaluable data for the end point analysis. Bacopa monnieri versus placebo significantly improved verbal learning as well as delayed recall as measured by the AVLT (p<.05). Though improvements were noted in the CFT, MAC-Q and TMT, there were no significant differences between placebo and active groups found for these tests. The Bacopa group reported a higher incidence of gastro-intestinal (GIT) side-effects than the placebo group, these predominantly being increased stool frequency, abdominal cramps and nausea. No other significant adverse effects were found. Conclusions A clinical trial was carried out to assess the effects of 12-weeks administration of Bacopa monnieri (300mg/day) on memory performance in people over the age of 55-years. Primary outcome measures were well validated neuropsychological tests that objectively measured verbal and visual memory and a memory complaint questionnaire that measured subjective memory complaints. The results demonstrated that Bacopa significantly improved memory acquisition and retention in older Australians. This concurs with findings from previous human and animal studies, as well as supports traditional Ayurvedic claims and uses. The beneficial effects on memory observed may be due to previously demonstrated antioxidant and cholinergic effects of the herb on the central nervous system. The use of Bacopa was associated with GIT side-effects, particularly increased bowel movements, nausea and abdominal cramping, findings infrequently reported previously. Possible explanations for these side-effects include GIT irritation by the saponin constituents of the herb, or cholinergic stimulation of autonomic and motor responses in the GIT, or a combination of both of these factors. The side-effects observed in the current study provide supportive evidence that Bacopa may increase cholinergic activity in humans. A worthwhile future extension of the current study would be to assess whether the finding of Bacopa’s efficacy for improving memory performance is replicable in populations with either mild cognitive impairment or early dementia.

Background This thesis investigated the efficacy and safety of Bacopa monnieri in improving memory in healthy Australians over the age of 55-years. A review of the literature showed that memory impairment and dementia are increasingly prevalent in the current demographic climate of an ageing population. As well as the pathological cognitive loss of neurodegenerative disease, many older persons are experiencing memory loss as part of the physiological process of ageing. Bacopa monnieri is a herbal medicine used since antiquity in the traditional Ayurvedic medical system of India for its cognitive enhancing effects. A number of pre-clinical and clinical studies support this traditional usage. Laboratory studies have demonstrated antioxidant and cholinergic actions in the brain as well as improved memory and cognitive performance in animal models. Human trials of Bacopa have also demonstrated improved memory performance. Some of these trials are limited by methodological flaws such as lack of blinding, small sample sizes, or use of outcome measurements which are not well validated. However, a small number of well designed human trials provide evidence for efficacy in cognitive and memory performance improvement. The current study was employed to extend on previous findings by assessing the efficacy and safety of Bacopa in the aged population specifically, as it is in this population that memory impairment becomes apparent. Aims 1. To assess the efficacy of Bacopa monnieri in improving memory in healthy Australians over the age of 55-years. 2. To assess whether the use of Bacopa is associated with side-effects Design A 12-week, randomised, double-blind, placebo-controlled, parallel group clinical trial. Participants Participants were self selected from the general population. They were aged 55-years or over at the commencement of the trial. Participants were without dementia, depression or other serious health conditions and did not use psychotropic medications. Intervention Participants were randomised to one of two treatment conditions, either a tableted extract of Bacopa monnieri called Bacomind™ (300mg/day, standardised to contain at least 40% bacosides), or an identical placebo. Participants attended three clinical evaluations: the first an initial screening session, the second a baseline evaluation of neuropsychological function and subjective memory performance at the commencement of the trial and the third, an end-of-trial outcome evaluation at 12-weeks, during which neuropsychological function and subjective memory performance were again assessed along with side-effects and study compliance. Primary Outcome Measures Rey Auditory Verbal Learning Test (AVLT), Rey-Osterrieth Complex Figure Test (CFT), Memory Complaint Questionnaire (MAC-Q), and Trail Making Test (TMT) Results From 136 people who elected to participate, 103 people met study entry criteria and 98 of these commenced the trial. Of these, 81 participants completed the trial and provided evaluable data for the end point analysis. Bacopa monnieri versus placebo significantly improved verbal learning as well as delayed recall as measured by the AVLT (p<.05). Though improvements were noted in the CFT, MAC-Q and TMT, there were no significant differences between placebo and active groups found for these tests. The Bacopa group reported a higher incidence of gastro-intestinal (GIT) side-effects than the placebo group, these predominantly being increased stool frequency, abdominal cramps and nausea. No other significant adverse effects were found. Conclusions A clinical trial was carried out to assess the effects of 12-weeks administration of Bacopa monnieri (300mg/day) on memory performance in people over the age of 55-years. Primary outcome measures were well validated neuropsychological tests that objectively measured verbal and visual memory and a memory complaint questionnaire that measured subjective memory complaints. The results demonstrated that Bacopa significantly improved memory acquisition and retention in older Australians. This concurs with findings from previous human and animal studies, as well as supports traditional Ayurvedic claims and uses. The beneficial effects on memory observed may be due to previously demonstrated antioxidant and cholinergic effects of the herb on the central nervous system. The use of Bacopa was associated with GIT side-effects, particularly increased bowel movements, nausea and abdominal cramping, findings infrequently reported previously. Possible explanations for these side-effects include GIT irritation by the saponin constituents of the herb, or cholinergic stimulation of autonomic and motor responses in the GIT, or a combination of both of these factors. The side-effects observed in the current study provide supportive evidence that Bacopa may increase cholinergic activity in humans. A worthwhile future extension of the current study would be to assess whether the finding of Bacopa’s efficacy for improving memory performance is replicable in populations with either mild cognitive impairment or early dementia.

As the battle against ever-increasing drug resistence bacteria rages on, novel and sometimes more complex natural products can be used to combat this. In this study, two-dimensional NMR techniques were utilized to collect a complete spectral data set for two natural products. The first structure, a synthesized Pyrrolobenzodiazepine alkaloid natural product was confirmed through these methods. The second, a strain of Rhodococcus, MTM3W5.2, produces a novel antibacterial molecule in broth cultures and the active compound was fractionated using a Sephedex LH-20 column. Chromatographic purification yielded a pure sample at 58.90 minutes, RT.58. HRMS data deduced an exact mass of 911.5490 Da, equivalent to a molecular formula of C52H78O13. Several major spin systems were constructed from the 2D-NMR spectra. However, due to limited sample quantity in compound with a large molecular weight and product instability, the long range HMBC signals needed to connect these fragments have not yet been obtained.

No description available.

An estimated 13.9% of Americans currently meet criteria for an alcohol (ethanol; EtOH) use disorder (AUD). While there are 4 medications approved by the Food and Drug Administration (FDA) to treat AUD, these treatments have demonstrated poor clinical efficacy. Our ongoing research program encompasses a multi-tiered screening of a natural product library and validation process to provide novel information about the mechanisms underlying EtOH-induced changes in neurobiology and to identify novel chemical scaffolds to be exploited in the development of pharmacological treatments for AUD in a rodent organotypic hippocampal slice culture model. Initial screens of several natural product compounds identified 3 compounds which attenuate 48 h EtOH-induced cytotoxicity in vitro. As analogs of natural products can be developed to have enhanced therapeutic potential over parental structures, Study 1 sought to extend on prior findings via the screening of several natural product analogs for their ability to attenuate EtOH-induced cytoxicity. Nine natural produce analogs demonstrated potent cytoprotective effects against EtOH-induced toxicity at 48 h. Several reports suggest EtOH-induced neurotoxicity may be secondary to the induction of persistent neuroimmune activation, and isoflavonoids have been shown to have effects on neuroimmune signaling. Thus, Study 2 compared the effects of compound 9b, an isoflavonoid analog identified in Study 1, to daidzein (DZ), a prototypical isoflavonoid, in the same 48 h model, with the addition of a neuroimmune component. Specifically, culture media was collected to assess for the release of the neuroimmune mediators HMGB1, TNF-α, IL-6, and IL-10 via ELISA. Compound 9b and DZ protected against EtOH-induced cytotoxicity at 48 h. EtOH exposure significantly increased secretion of HMGB1 and IL-6 into culture media at 48h. Compound 9b and DZ attenuated these increases at all concentrations tested. These results suggest potential neuroimmune modulating properties of isoflavonoids which may contribute to their neuroprotective effects against EtOH in vitro. These findings highlight the potential applications DZ and the novel isoflavonoid analog 9b for use in the treatment of AUD.

Introduction: Genetic rearrangements in Ewing sarcoma, prostate, and leukemia cells result in activation of oncogenic ETS transcription factor fusions. Mithramycin (MTM) has been identified as an inhibitor of EWS-FLI1 transcription factor, a gene fusion product responsible for oncogenesis in Ewing sarcoma. Despite preclinical success, a phase I/II clinical trial testing MTM therapy in refractory Ewing sarcoma was terminated. Liver and blood toxicities resulted in dose de-escalation and sub-therapeutic exposures. However, the promise of selectively targeting oncogenic ETS transcription factors like EWS-FLI1 prompted us to undertake the discovery of more selective, less toxic analogues of MTM. MTM is a potent inhibitor of ubiquitous SP1 transcription factor, likely inducing non-specific toxicity. In collaboration with two medicinal chemistry groups, two semi-synthetic efforts were implemented to develop novel analogues of MTM. The first effort utilized the biosynthetic product mithramycin SA (MTMSA) to modify C3-side chain. The second effort utilized an oxime linker directly formed on MTM’s C3-side chain (MTM-oxime; MTMox). Here I present the pharmacological assessment of over 75 novel MTM analogues towards selectively targeting oncogenic ETS transcription factors, like EWS-FLI1, over ubiquitous transcription factors, like SP1. Methods: Novel MTM analogues were evaluated for selective cytotoxicity against ETS fusion-dependent cell lines. Selectively cytotoxic analogues were evaluated for inhibitory effects on several gene promoters in TC-32 reporter cell lines, a Ewing sarcoma cell line dependent on EWS-FLI1, transfected with luciferase reporter vector. Cloned reporter vectors incorporated NR0B1 (EWS-FLI1 binding), β-actin (SP1 binding) and CMV (non-specific) gene promoters. Furthermore, gene (mRNA) and protein expression changes of EWS-FLI1 and SP1, as well as regulated target genes, namely NR0B1, VEGFA and BCL-2 were evaluated with MTM analogue treatments. The MTM analogues with most selective activity in vitro were administered to mice by intravenous bolus dose for pharmacokinetic analysis. The MTM analogues with highest systemic exposure from each semi-synthetic effort, namely MTMSA-Trp-A10 and MTMox-24, were further evaluated. Metabolic stabilities in whole blood, plasma, and tumor cell matrices, and across multiple species were compared with MTM. Moreover, intrinsic hepatic clearances were estimated using mouse liver microsomes. Tumor and liver distributions were estimated in tumor bearing mice. Additionally, the effect of organic anionic transporter polypeptides (OATP) on distribution of MTM was investigated. Maximum tolerated doses were evaluated for lead MTM analogues, having both selective activities in vitro and high systemic exposure, compared to MTM. Complete blood cell counts and plasma alanine aminotransferase activity were measured to evaluate dose-dependent blood and liver toxicities, respectively. ETS fusion-dependent and non-dependent cell lines were implanted subcutaneously into immunocompromised mice for efficacy studies. Average tumor volumes and survival were tracked for mice receiving treatment, compared to MTM and vehicle treatment. Results: Evaluation of MTM analogues from both semi-synthetic efforts revealed that conjugation of MTM C3-side chain with tryptophan (Trp) and/or phenylalanine (Phe) improved selective cytotoxicity against ETS fusion-dependent cell lines. This was highlighted by MTMSA-Trp-A2 (also refer to as MTMSA-Phe-Trp) and MTMSA-Trp-A10 (also refer to as MTMSA-Trp-Trp), with selective indices of 19.1 and 15.6, respectively, compared to MTM (1.5). Similarly, MTMox-23 (also refer to as MTMox-Phe-Trp) and MTMox-20 (also refer to as MTMox-Trp) had selectivity indices of 4.6 and 4.5, respectively. These selectively cytotoxic MTM analogues inhibited EWS-FLI1-mediated transcription 10-fold more effectively than both non-specific CMV-mediated and SP1-mediated (via β-actin promoter) transcription in TC-32 reporter cell lines. Moreover, gene (mRNA) and protein expression of EWS-FLI1 and regulated gene, NR0B1, were inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Similarly, SP1 and target genes, VEGFA and BCL-2, gene (mRNA) and protein expressions were also inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Conjugation of Trp and/or Phe to C3-side chain of MTM increased systemic exposure in vivo. Most impressively, the addition of two Trp residues, namely MTMSA-Trp-A10 and MTMox-24 (also refer to as MTMox-Trp-Trp), resulted in systemic exposure increases of 218- and 42-fold, respectively, after intravenous (IV) bolus dose. Metabolically, tryptophan/phenylalanine conjugated MTM analogues are liable to esterase activity on carboxy-methyl functional group. Very rapid de-methylation in biological matrix was observed with MTMox-24, compared to MTMSA-Trp-A10, suggesting a regiospecific effect. However, esterase activity was limited to rodent matrices and demethylation occurred at significantly diminished rates in non-human primate and human plasma. MTM analogues were not susceptible to p450-mediated metabolism, with negligible loss in mouse liver microsome assay compared to verapamil control. MTM (1mg/kg) and MTMox-24 (6mg/kg) were detected in subcutaneously implanted (flank) LL2 tumors and liver homogenates after IV bolus dose. Interestingly, MTMSA-Trp-A10 (2mg/kg) was not. Despite a 3-fold increase in systemic exposure with rifampin oral pretreatment, an OATP inhibitor, exposure of MTM was unaffected in Oatp knockout mouse model. Exposure of MTM in liver tissue was 8.4-fold higher compared to tumor tissue with low tissue clearance. This agrees with the lack of metabolism observed in liver microsomes and may provide a mechanism for clinically observed liver toxicity. MTMSATrp-A10 had a single maximum tolerated dose (MTD) of 0.75mg/kg, compared to 1mg/kg for MTM, administered by IV bolus. In contrast, MTM-oxime analogues (MTMox-20, -23, -24 and -25) had single maximum tolerated doses of 20 – 25mg/kg. These increased tolerances are the result of additive differences in whole blood stability, cytotoxicity and systemic exposure. At a dose of 0.75mg/kg, administered every 3 days, MTMSA-Trp-A10 did not result in an efficacious result in tumor xenograft studies. These studies remain under further investigation, but the result may indicate high plasma protein binding of MTMSA-Trp-A10 and lack of free fraction available within tumor. The most selective MTM-oxime analogue in vitro, MTMox-23, significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0025, day 16, one-way ANOVA multiple comparisons test) compared to MTM (p=0.1174, day 16) and extending survival for 17 days out of 48 days on study (p=0.0003, Log Rank (Mantel-Cox) single comparison test) with treatment at MTD every 3 days, compared to vehicle. Additionally, the MTM-oxime analogue with highest systemic exposure, MTMox-24, also significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0003, day 21, one-way ANOVA multiple comparisons test) compared to MTM (p=0.032, day 21) and extending survival for 12 days out of 37 days on study (p=0.0004, Log Rank (Mantel-Cox) single comparison test) with treatment, compared to vehicle. Conclusion: These studies in whole highlight the importance of exposure (pharmacokinetics; PK), toxicity and efficacy (pharmacodynamics; PD) relationships. The cytotoxicity and high systemic exposure of MTMSA-Trp-A10 directly contributes to its lower tolerated dose. However, despite a similar tolerated dose to MTM, systemic exposure remains 163-fold higher at the MTD. High systemic exposure may be attributed to high plasma protein binding, but also reduces the exposure of free MTMSA-Trp-A10 within the tumor tissue, which drives the efficacious response. In contrast, the less cytotoxic and rapidly de-methylated MTM-oxime analogues allow for 25-fold higher tolerances in mice. This unique metabolism and clearance may prevent exposures required to induced systemic blood and liver toxicities induced by MTM. Moreover, at these highly tolerated doses, the initial systemic exposure at MTD is highest among analogues tested, which resulted in an efficacious response with MTMox-23 and MTMox-24 treatment in tumor xenograft models. It remains to be determined if these PK/PD relationships can be reproduced in additional animal models, including human, without inducing toxicity. Nonetheless, these initial studies in mice demonstrate that a more selective, more tolerated analogue of MTM has potential for clinical success in treating ETS fusion-dependent tumors.

Microbial natural products have been a source for new drugs for many decades and are unrivaled in their capacity to generate not only future therapeutic agents, but also providing key agents for agricultural and industrial use. LC-MS/MS based metabolomic tools and technologies have been developed that can rapidly dereplicate nonribosomal peptides and statistically identify related congeners in an automated nontargeted process from complex natural product extracts with nanogram sensitivity. This data-base search approach is designed to handle linear, cyclic and cyclic-branched nonribosomal peptides from proteinogenic and nonproteinogenic amino acids without genomic data or traditional bioactivity directed fractionation. Chemometric work-flows combined with a comprehensive metabolomic guided discovery strategy were used to profile the chemical space of a diverse collection of understudied fungal endophytes from fruiting plants. This approach allowed for the prioritization of unique isolates and for the focused discovery, isolation and characterization of distinct outlier metabolites by LC-SPE, 1D and 2D NMR, HRMS and single crystal X-ray analysis. These metabolomic tools and strategies have led to the discovery and characterization of 35 new and over 40 known natural products, many of which are biologically active. This thesis with enabling metabolomic tools and novel discoveries has demonstrated the utility of these analytical methodologies as an effective strategy for the untargeted discovery of new natural products from bacteria and endophytic fungi. / Thesis / Doctor of Philosophy (PhD)